Freeze-dried product and gas-filled microvesicles suspension

ABSTRACT

A method of manufacturing a suspension of gas-filled microvesicles by reconstituting a freeze-dried product and a suspension obtained according to said method, where the freeze-dried product has been subjected to a thermal treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national stage application of correspondinginternational application number PCT/EP2020/063559, filed May 14, 2020,which is a continuation of U.S. application Ser. No. 16/788,083, filedFeb. 11, 2020, which is a continuation-in-part of U.S. application Ser.No. 16/688,540, filed Nov. 19, 2019, which is a continuation-in-part ofU.S. application Ser. No. 16/413,526, filed May 15, 2019, which arehereby incorporated by reference in their entireties.

TECHNICAL FIELD

The invention relates to a new method of manufacturing a suspension ofgas-filled microvesicles by reconstituting a freeze-dried product and tothe suspension obtained according to said method.

BACKGROUND OF THE INVENTION

Rapid development of contrast agents in the recent years has generated anumber of different compositions and formulations, which are useful incontrast-enhanced imaging of organs and tissues of human or animal bodyas well as in therapeutic treatments thereof.

A class of contrast agents particularly useful for Contrast EnhancedUltraSound imaging (“CEUS” imaging) includes suspensions of gas bubblesof nano- and/or micro-metric size dispersed in an aqueous medium. Thegas is typically entrapped or encapsulated in a film-layer comprising,for instance, emulsifiers, oils, thickeners or sugars. These stabilizedgas bubbles (dispersed in a suitable physiological solution) aregenerally referred to in the art with various terminologies, dependingtypically from the stabilizing material employed for their preparation;these terms include, for instance, “microspheres”, “microbubbles”,“microcapsules” or “microballoons”, globally referred to here as“gas-filled microvesicles” (or “microvesicles”).

UltraSound Contrast Agents (“USCAs”) can be produced according tovarious manufacturing methods. One of these methods, see e.g.WO94/09829, entails the dissolution of an amphiphilic material (such asa phospholipid and/or fatty acid) and of a freeze-drying protectingcompound (e.g. polyetheleneglycol) in an organic solvent; the obtainedmixture is then subjected to freeze-drying, typically after being filledinto vials, to remove the solvent and obtain a freeze-dried product.Another method, see e.g. WO2004/069284, entails the preparation of amicroemulsion of water with a water immiscible organic solvent, saidemulsion comprising an amphiphilic material and a freeze-dryingprotecting compound. The emulsion is then and subjected (upondistribution into vials) to a freeze-drying step to remove water andsolvent.

The headspace of the vials, containing a freeze-dried solid product inpowder form at the bottom thereof, is then filled with a suitable gas(e.g. a fluorinated gas) and finally sealed for storage. Before use, anaqueous suspension of microbubbles is easily prepared by introducing asuitable liquid into the vial (e.g. saline) and gently shaking the vialto dissolve the freeze-dried product.

A commercially available USCA which can be manufactured according to theabove method is SonoVue® (or Lumason® in the USA), from Bracco.

The Applicant has now observed that the characteristics of a suspensionof gas-filled microvesicles (particularly microbubbles) reconstitutedfrom a freeze-dried product can be improved by introducing a finalcontrolled thermal treatment (i.e. heating) at the end of the processfor manufacturing the freeze-dried solid product.

SUMMARY OF THE INVENTION

According to an aspect, the invention relates to a method ofmanufacturing a freeze-dried composition suitable for the preparation ofa suspension of stabilized gas-filled microvesicles, said compositioncomprising: (i) an amphiphilic material capable of stabilizing said gasmicrovesicles; and (ii) a freeze-drying protecting component; whichcomprises:

-   -   a. preparing a liquid mixture comprising said amphiphilic        material and said freeze-drying protecting component in a        solvent;    -   b. freeze-drying the liquid mixture to remove said solvent and        obtain a freeze-dried product comprising said amphiphilic        material and said freeze-drying protecting component; and    -   c. heating said freeze-dried product.

Preferably, said heating step comprises heating said product at atemperature higher than 35° C., more preferably at least 38° C. Theheating temperature is preferably lower than 50° C., more preferablylower than 48° C.

In certain embodiments, the heating step is performed at ambientpressure.

Preferably, the heating step lasts for at least 8 hours, more preferablyfor at least 12 hours.

According to another aspect, the invention relates to a freeze-driedproduct obtained according to the manufacturing method described above.

According to another aspect, the invention relates to a suspension ofgas-filled microvesicles obtained by reconstituting a freeze-driedproduct prepared according to the method of manufacturing describedabove, said suspension being obtained by admixing said product with apharmaceutically acceptable liquid carrier in the presence of aphysiologically acceptable gas under gentle agitation.

According to a further aspect the invention relates to method formanufacturing a suspension of gas-filled microvesicles stabilized by anamphiphilic material, which comprises:

-   -   a. preparing a freeze-dried product according to the        manufacturing method illustrated above; and    -   b. reconstituting said product by admixing it with a        pharmaceutically acceptable liquid carrier in the presence of a        physiologically acceptable gas under gentle agitation, to obtain        the suspension of gas-filled microvesicles.

DETAILED DESCRIPTION OF THE INVENTION

A suitable method for preparing injectable suspensions of gas-filledmicrovesicles comprises the reconstitution, in the presence of asuitable physiologically acceptable gas, of a freeze-dried productcomprising an amphiphilic material capable of stabilizing saidmicrovesicles (e.g. by forming a stabilizing layer at the liquid-gasinterface) with an aqueous carrier.

The freeze-dried product is typically obtained by freeze-drying a liquidmixture comprising said amphiphilic material and a freeze-dryingprotecting component in a suitable solvent.

The liquid mixture which undergoes the freeze-drying process can beobtained according methods know in the art, disclosed e.g. in WO94/09829or WO2004/069284.

Preparation of Liquid Mixture for Freeze-Drying

For instance, according to the process disclosed by WO94/09829, theamphiphilic material is dispersed into an organic solvent (e.g. tertiarybutanol, dioxane, cyclohexanol, tetrachlorodifluoro ethylene or2-methyl-2-butanol) together with a suitable freeze-drying protectingcomponent. The dispersion containing the amphiphilic material and thefreeze-drying protecting component is then subjected to freeze-drying toremove the organic solvent thus obtaining a freeze-dried product.

According to the alternative process disclosed in WO2004/069284, acomposition comprising an amphiphilic material may be dispersed in anemulsion of water with a water immiscible organic solvent underagitation, preferably in admixture with a freeze-drying protectingcomponent.

Suitable water immiscible organic solvents include, for instance,branched or linear alkanes, alkenes, cyclo-alkanes, aromatichydrocarbons, alkyl ethers, ketones, halogenated hydrocarbons,perfluorinated hydrocarbons or mixtures thereof.

The emulsion may be obtained by submitting the aqueous medium and thesolvent, in the presence of the amphiphilic material, to any appropriateemulsion-generating technique known in the art such as, for instance,sonication, shaking, high pressure homogenization, micromixing, membraneemulsification, high speed stirring or high shear mixing. Thefreeze-drying protecting component can be added either before or afterthe formation of the emulsion, e.g. as an aqueous solution comprisingsuch freeze-drying protecting component. The so obtained microemulsion,which contains microdroplets of solvent surrounded and stabilized by theamphiphilic material, is then freeze-dried according to conventionaltechniques to obtain a freeze-dried material, which can then be used forpreparing a suspension of gas-filled microvesicles.

Amphiphilic Material

According to a preferred embodiment, amphiphilic materials useful forpreparing the above liquid mixtures comprise a phospholipid.Phospholipids, as other amphiphilic molecules, are generally capable offorming a stabilizing film of material (typically in the form of amono-molecular layer) at the gas-water boundary interface in the finalgas-filled microvesicles suspension, these materials are also referredto in the art as “film-forming” materials.

Phospholipids typically contain at least one phosphate group and atleast one, preferably two, lipophilic long-chain hydrocarbon group.

Examples of suitable phospholipids include esters of glycerol with oneor preferably two (equal or different) residues of fatty acids and withphosphoric acid, wherein the phosphoric acid residue is in turn bound toa hydrophilic group, such a, for instance, choline(phosphatidylcholines—PC), serine (phosphatidylserines—PS), glycerol(phosphatidylglycerols—PG), ethanolamine (phosphatidylethanolamines—PE),inositol (phosphatidylinositol). Esters of phospholipids with only oneresidue of fatty acid are generally referred to in the art as the “lyso”forms of the phospholipid or “lysophospholipids”. Fatty acids residuespresent in the phospholipids are in general long chain aliphatic acids,typically containing from 12 to 24 carbon atoms, preferably from 14 to22; the aliphatic chain may contain one or more unsaturations or ispreferably completely saturated. Examples of suitable fatty acidsincluded in the phospholipids are, for instance, lauric acid, myristicacid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleicacid, linoleic acid, and linolenic acid. Preferably, saturated fattyacids such as myristic acid, palmitic acid, stearic acid and arachidicacid are employed.

Further examples of phospholipid are phosphatidic acids, i.e. thediesters of glycerol-phosphoric acid with fatty acids; sphingolipidssuch as sphingomyelins, i.e. those phosphatidylcholine analogs where theresidue of glycerol diester with fatty acids is replaced by a ceramidechain; cardiolipins, i.e. the esters of 1,3-diphosphatidylglycerol witha fatty acid; glycolipids such as gangliosides GM1 (or GM2) orcerebrosides; glucolipids; sulfatides and glycosphingolipids.

As used herein, the term “phospholipid(s)” includes either naturallyoccurring, semisynthetic or synthetically prepared compounds that can beemployed either singularly or as mixtures.

Examples of naturally occurring phospholipids are natural lecithins(phosphatidylcholine (PC) derivatives) such as, typically, soya bean oregg yolk lecithins.

Examples of semisynthetic phospholipids are the partially or fullyhydrogenated derivatives of the naturally occurring lecithins. Preferredphospholipids are fatty acids di-esters of phosphatidylcholine,ethylphosphatidylcholine, phosphatidylglycerol, phosphatidic acid,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol or ofsphingomyelin.

Examples of preferred phospholipids are, for instance,dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine(DMPC), dipalmitoyl-phosphatidylcholine (DPPC),diarachidoyl-phosphatidylcholine (DAPC), distearoyl-phosphatidylcholine(DSPC), dioleoyl-phosphatidylcholine (DOPC), 1,2Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC),dipentadecanoyl-phosphatidylcholine (DPDPC),1-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC),1-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC),1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC),1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC),1-palmitoyl-2-oleylphosphatidylcholine (POPC),1-oleyl-2-palmitoyl-phosphatidylcholine (OPPC),dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts,diarachidoylphosphatidyl-glycerol (DAPG) and its alkali metal salts,dimyristoylphosphatidylglycerol (DMPG) and its alkali metal salts,dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts,distearoylphosphatidylglycerol (DSPG) and its alkali metal salts,dioleoyl-phosphatidylglycerol (DOPG) and its alkali metal salts,dimyristoyl phosphatidic acid (DMPA) and its alkali metal salts,dipalmitoyl phosphatidic acid (DPPA) and its alkali metal salts,distearoyl phosphatidic acid (DSPA), diarachidoylphosphatidic acid(DAPA) and its alkali metal salts, dimyristoyl-phosphatidylethanolamine(DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidyl-ethanolamine (DSPE), dioleylphosphatidyl-ethanolamine(DOPE), diarachidoylphosphatidyl-ethanolamine (DAPE),dilinoleylphosphatidylethanolamine (DLPE), dimyristoylphosphatidylserine (DMPS), diarachidoyl phosphatidylserine (DAPS),dipalmitoyl phosphatidylserine (DPPS), distearoylphosphatidylserine(DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoyl sphingomyelin(DPSP), and distearoylsphingomyelin (DSSP),dilauroyl-phosphatidylinositol (DLPI), diarachidoylphosphatidylinositol(DAPI), dimyristoylphosphatidylinositol (DMPI),dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol(DSPI), dioleoyl-phosphatidylinositol (DOPI).

Suitable phospholipids further include phospholipids modified by linkinga hydrophilic polymer, such as polyethyleneglycol (PEG) orpolypropyleneglycol (PPG), thereto. Preferred polymer-modifiedphospholipids include “pegylated phospholipids”, i.e. phospholipidsbound to a PEG polymer. Examples of pegylated phospholipids arepegylated phosphatidylethanolamines (“PE-PEGs” in brief) i.e.phosphatidylethanolamines where the hydrophilic ethanolamine moiety islinked to a PEG molecule of variable molecular weight (e.g. from 300 to20000 daltons, preferably from 500 to 5000 daltons), such as DPPE-PEG(or DSPE-PEG, DMPE-PEG, DAPE-PEG or DOPE-PEG). For example, DPPE-PEG5000refers to DPPE having attached thereto a PEG polymer having a meanaverage molecular weight of about 5000. An example of DPPE-PEG5000 isthe methoxy terminated PEG derivative1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000].

In an embodiment the phospholipids may bear a reactive moiety which maythen be reacted with a corresponding reactive moiety bearing a suitableactive component (e.g. targeting ligand), in order to bind said activecomponent to the microvesicle. Examples of suitable reactive moietiesinclude, for instance, reactive groups capable of reacting with an aminogroup bound to an active component such as isothiocyanate groups (thatwill form a thiourea bond), reactive esters (to form an amide bond),aldehyde groups (for the formation of an imine bond to be reduced to analkylamine bond); reactive groups capable of reacting with a thiol groupbound to an active component, such as haloacetyl derivatives ormaleimides (to form a thioether bond); reactive groups capable ofreacting with a carboxylic group bound to an active component, such asamines or hydrazides (to form amide or alkylamide bonds). Preferably,the amphiphilic compound bearing the reactive moiety is a lipid bearinga hydrophilic polymer, such as those previously mentioned, preferably apegylated phospholipid, e.g. DPPE-PEG2000, such asDPPE-PEG2000-maleimide.

Particularly preferred phospholipids are DAPC, DSPC, DPPC, DMPA, DPPA,DSPA, DMPG, DPPG, DSPG, DMPS, DPPS, DSPS and Ethyl-DSPC. Most preferredare DPPG, DPPS and DSPC.

Mixtures of phospholipids can also be used, such as, for instance,mixtures of DPPE and/or DSPE (including pegylated derivates), DPPC, DSPCand/or DAPC with DSPS, DPPS, DSPA, DPPA, DSPG, DPPG, Ethyl-DSPC and/orEthyl-DPPC.

The phospholipids can conveniently be used in admixture with any othercompound, preferably amphiphilic. For instance, lipids such ascholesterol, ergosterol, phytosterol, sitosterol, lanosterol,tocopherol, propyl gallate or ascorbyl palmitate, fatty acids such asmyristic acid, palmitic acid, stearic acid, arachidic acid andderivatives thereof or butylated hydroxytoluene and/or othernon-phospholipid (amphiphilic) compounds can optionally be added to oneor more of the foregoing phospholipids, e.g. in proportions preferablybelow 50% by weight, more preferably up to 25% or lower. Particularlypreferred as additional compound in admixture with phospholipids arefatty acids. Fatty acids useful in a composition according to theinvention, which can be either saturated or unsaturated, comprise aC₁₀-C₂₄, aliphatic chain terminated by a carboxylic acid moiety,preferably a C₁₄-C₂₂ and more preferably a C₁₆-C₂₀ aliphatic chain.Examples of suitable saturated fatty acids include capric (n-decanoic),lauric (n-dodecanoic), myristic (n-tetradecanoic), palmitic(n-hexadecanoic), stearic (n-octadecanoic), arachidic (n-eicosanoic),behenic (n-docosanoic) and n-tetracosanoic acid. Preferred saturatedfatty acids are myristic, palmitic, stearic and arachidic acid, morepreferably palmitic acid. Examples of unsaturated fatty acids comprisemyristoleic (cis-9-tetradecenoic), palmitoleic (cis-9-hexadecenoic),sapienic (cis-6-hexadecenoic), oleic (cis-9-octadecenoic), linoleic(cis-9,12-octadecadienoic), linolenic (cis-9,12,15-octadecatrienoic),gondoic (cis-11-eicosenoic), cis-11,14-eicosadienoic,cis-5,8,11-eicosatrienoic, cis-8,11,14-eicosatrienoic,cis-11,14,17-eicosatrienoic, arachidonic(cis-8,11,14,17-eicosatetraenoic) and erucic (cis-13-docosenoic) acid.

According to an embodiment, the mixture of amphiphilic materialscomprises a mixture of DSPC, DPPG and palmitic acid.

According to an alternative embodiment, said amphiphilic materialcomprises a mixture of DSPC, DPPE-PEG5000 and palmitic acid, optionallyfurther comprising a targeting ligand

Targeting Ligands

Compositions and microvesicles according to the invention may optionallycomprise a targeting ligand.

The term “targeting ligand” includes within its meaning any compound,moiety or residue having, or being capable to promote, a targetingactivity (e.g. including a selective binding) of the microvesicles of acomposition of the invention towards any biological or pathological sitewithin a living body. Targets with which targeting ligand may beassociated include tissues such as, for instance, myocardial tissue(including myocardial cells and cardiomyocytes), membranous tissues(including endothelium and epithelium), laminae, connective tissue(including interstitial tissue) or tumors; blood clots; and receptorssuch as, for instance, cell-surface receptors for peptide hormones,neurotransmitters, antigens, complement fragments, and immunoglobulinsand cytoplasmic receptors for steroid hormones.

The targeting ligand may be synthetic, semi-synthetic, ornaturally-occurring. Materials or substances which may serve astargeting ligands include, for example, but are not limited to proteins,including antibodies, antibody fragments, receptor molecules, receptorbinding molecules, glycoproteins and lectins; peptides, includingoligopeptides and polypeptides; peptidomimetics; saccharides, includingmono and polysaccharides; vitamins; steroids, steroid analogs, hormones,cofactors, bioactive agents and genetic material, including nucleosides,nucleotides and polynucleotides.

The targeting ligand may be a compound per se which is admixed with theother components of the microvesicle or may be a compound which is boundto an amphiphilic molecule (typically a phospholipid) employed for theformation of the microvesicle.

In one preferred embodiment, the targeting ligand may be bound to anamphiphilic molecule (e.g. a phospholipid) forming the stabilizingenvelope of the microvesicle through a covalent bond. In such a case,the specific reactive moiety that needs to be present on the amphiphilicmolecule will depend on the particular targeting ligand to be coupledthereto, as illustrated in detail above. In order to covalently bind adesired targeting ligand, at least part of the amphiphilic compoundforming the microvesicle's envelope shall thus contain a suitablereactive moiety and the targeting ligand containing the complementaryfunctionality will be linked thereto according to known techniques, e.g.by adding it to a dispersion comprising the amphiphilic components ofthe microvesicle. Preferably, the amphiphilic compound is a lipidbearing a hydrophilic polymer, such as those previously mentioned,preferably a pegylated phospholipid (e.g. DSPE-PEG2000). In this case,the targeting ligand is linked to a suitable reactive moiety on thehydrophilic polymer (e.g. DSPE-PEG2000-NH₂), optionally through alinker. The amphiphilic compound may be combined with the desiredtargeting ligand before preparing the microvesicle, and the so obtainedcombination may be used for the preparation of the microvesicle.Alternatively, the targeting ligand may be linked to the respectiveamphiphilic compound during the preparation of the microvesicle (e.g. inthe intermediate microemulsion preparation of the process described inWO2004/069284). As a further alternative, the binding may take place onthe formed microvesicle comprising an amphiphilic material bearing areactive moiety.

According to an alternative embodiment, the targeting ligand may also besuitably associated with the microvesicle via physical and/orelectrostatic interaction. As an example, a functional moiety having ahigh affinity and selectivity for a complementary moiety may beintroduced into the amphiphilic molecule, while the complementary moietywill be linked to the targeting ligand. For instance, an avidin (orstreptavidin) moiety (having high affinity for biotin) may be covalentlylinked to a phospholipid (or to a pegylated phospholipid) while thecomplementary biotin moiety may be incorporated into a suitabletargeting ligand, e.g. a peptide or an antibody. The biotin-labelledtargeting ligand will thus be associated with the avidin-labelledphospholipid of the microvesicle by means of the avidin-biotin couplingsystem. Alternatively, both the phospholipid and the targeting ligandmay be provided with a biotin moiety and subsequently coupled to eachother by means of avidin (which is a bifunctional component capable ofbridging the two biotin moieties). Examples of biotin/avidin coupling ofphospholipids and peptides are also disclosed in the above cited U.S.Pat. No. 6,139,819. Alternatively, van der Waal's interactions,electrostatic interactions and other association processes may associatewith or bind to the targeting ligand to the amphiphilic molecules.

Alternatively, the phospholipid may be modified with a protein suitablefor specific coupling to Fc domain of Immunoglubulin (Ig) such asProtein A, Protein G, Protein A/G or Protein L. According to analternative embodiment, the targeting ligand may be a compound which isadmixed with the components forming the microvesicle, to be eventuallyincorporated the microvesicle structure, such as, for instance, alipopeptide as disclosed e.g. in International patent Applications WO98/18501 or 99/55383.

Alternatively, a microvesicle may first be manufactured, which comprisesa compound (lipid or polymer-modified lipid) having a suitable moietycapable of interacting with a corresponding complementary moiety of atargeting ligand; thereafter, the desired targeting ligand is added tothe microvesicle suspension, to bind to the corresponding complementarymoiety on the microvesicle.

Examples of suitable specific targets to which the microvesicles may bedirected are, for instance, fibrin and the GPIIbIIIa binding receptor onactivated platelets. Fibrin and platelets are in fact generally presentin “thrombi”, i.e. coagula which may form in the blood stream and causea vascular obstruction. Suitable binding peptides are disclosed, forinstance, in the above cited U.S. Pat. No. 6,139,819. Further bindingpeptides specific for fibrin-targeting are disclosed, for instance, inInternational patent application WO 02/055544.

Other examples of important targets include receptors in vulnerableplaques and tumor specific receptors, such as kinase domain region (KDR)and VEGF (vascular endothelial growth factor)/KDR complex. Bindingpeptides suitable for KDR or VEGF/KDR complex are disclosed, forinstance, in International Patent application WO 03/74005, WO 03/084574and WO2007/067979. In an embodiment, the targeting peptide is a dimericpeptide-phospholipid conjugate (lipopeptide) as described inWO2007/067979.

Freeze-Drying Protecting Component

As defined herein, a freeze-drying protecting component is a compoundwith cryoprotective and/or lyoprotective effect. Suitable freeze-dryingprotecting components include, for instance, carbohydrates, e.g. a mono-di- or poly-saccharide, such as sucrose, maltose, trehalose, glucose,lactose, galactose, raffinose, cyclodextrin, dextran, chitosan and itsderivatives (e.g. carboxymethyl chitosan, trimethyl chitosan); polyols,e.g. sugar alcohols such as sorbitol, mannitol or xylitol; orhydrophilic polymers, e.g. polyoxyalkyleneglycol such as polyethyleneglycol (e.g. PEG2000, PEG4000 or PEG8000) or polypropylenglycol.According to an embodiment said freeze-drying protecting component ispolyethylene glycol, preferably PEG4000. PEG4000 as used herein has itsnormal meaning in the field, indicating a polyethyleneglycol having amolecular weight of about 4000 g/mole, in general with a variation of+/−10% around said value.

Freeze-Drying Process

For the freeze-drying process, the liquid mixture containing theamphiphilic material and the freeze-drying protecting component(obtained e.g. according to either of the previously illustratedmanufacturing processes), is typically sampled into glass vials (e.g.DIN8R or DIN20R) which are loaded into a freeze-dryer.

The freeze-drying process generally includes an initial step (primarydrying) where the vials are rapidly deep-cooled (e.g. at temperatures offrom −35° C. to −70° C.) to freeze the liquid(s) of the mixture and thensubjected to vacuum (e.g. 0.1-0.8 mbar); during the primary drying, thesubstantial totality of the frozen liquid(s) (e.g. water and/orsolvents) is removed by sublimation, typically up to about 95% of thetotal amount of liquid, preferably up to about 99%. After the primarydrying, residual liquid (including possible interstitial water) can befurther removed during the secondary drying, which is typicallyconducted at a temperature higher than room temperature, under vacuum(preferably by maintaining the same vacuum applied during the primarydrying). The temperature during the secondary drying is preferably nothigher than 35° C. The secondary drying can be stopped when the residualcontent of the liquid(s) reaches a desired minimum value, e.g. less 3%(preferably less than 1%) by weight of water with respect to the totalmass of residual freeze-dried product, or e.g. less than 0.01% byweight, preferably less than 0.08%, for residual solvent(s).

After completion of the freeze-drying process (i.e. stopping of heatingand vacuum removal), the freeze-dried product can undergo the additionalthermal treatment step according to the invention, under ambientpressure. As used herein, the term “ambient pressure” refers to thenormal value of atmospheric pressure values (i.e. about 103.12 kPa atsea level, typically between 95 and 104 kPa). Preferably the thermaltreatment is performed on the sealed vial, after saturating theheadspace of the vials containing the freeze-dried product with asuitable physiologically acceptable gas and then stoppering (e.g. with arubber, such as butyl rubber, stopper) and sealing (e.g. with a metal,such as aluminium, crimp seal) the vials. In this case, the vials arepreferably removed from the freeze-drier and introduced in a suitableoven for the thermal treatment. Alternatively, such thermal treatmentcan be performed on the open vial (which is preferably kept into thefreeze-dryer), which is then saturated with the gas and thenstoppered/sealed.

Examples of suitable physiologically acceptable gases include, forinstance, fluorinated gases such as SF₆, C₃F₈, C₄F₁₀, optionally inadmixture with air or nitrogen.

In an embodiment, the gas SF₆ is used in combination with a mixture ofamphiphilic materials comprising DSPC, DPPG and palmitic acid, as abovedefined.

In another embodiment, the gas C₄F₁₀, or a mixture of C₄F₁₀ withnitrogen, is used in combination with a mixture of amphiphilic materialscomprising DSPC, DPPE-PEG5000 and palmitic acid, optionally furthercomprising a targeting ligand, as above defined

Other Components

Other components, e.g. excipients or additives, may either be present inthe dry formulation for the preparation of the microvesicles or may beadded together with the aqueous carrier used for the reconstitutionthereof, without necessarily being involved (or only partially involved)in the formation of the stabilizing envelope of the microvesicle. Theseinclude for instance pH regulators, osmolality adjusters, viscosityenhancers, emulsifiers, bulking agents, etc. and may be used inconventional amounts.

Suspension of Gas-Filled Microvesicles

The suspension of gas-filled microvesicles can then be prepared byreconstituting the freeze-dried product with a physiologicallyacceptable (aqueous) carrier, under gentle agitation. Suitablephysiologically acceptable (aqueous) carriers include, for instance,water for injection, saline or glucose solution, optionally containingexcipients or additives as illustrated above.

Heat Treatment

According to the invention, the freeze-dried product (contained inrespective vials at the end of the freeze-drying process) advantageouslyundergoes an additional final step of heat treatment (or thermaltreatment).

As mentioned before, the thermal treatment is preferably performed onthe freeze-dried product in the sealed vials already containing thephysiologically acceptable gas; alternatively, it can be performed onthe freeze-dried product in the vials before filling them with the gasand sealing. In the first case the thermal treatment can be eitheraccomplished within the freeze-drier apparatus or preferably in aseparate heating device (e.g. an oven). In the second case the heatingstep is preferably performed within the lyophilizing apparatus;afterward, the atmosphere is saturated with the desired gas and thevials are sealed.

As observed by the Applicant, said heat treatment of the freeze-driedproduct surprisingly results in improved characteristics of thesuspension of gas-filled microvesicles obtained upon reconstituting ofthe freeze-dried product, with respect to suspensions obtained fromfreeze-dried products which do not undergo such heat treatment.

Applicant observed in particular that such treatment results in anincreased resistance to pressure of the obtained microvesicles.

The freeze-dried product is preferably heated at a temperature higherthan 35° C. (e.g. 36° C.), more preferably at a temperature of 38° C. orhigher. The maximum temperature of the heat treatment generally dependson the materials comprised in the freeze-dried product. For instance,such temperature shall be lower than the melting point of the materialused as freeze-drying additive, which is the component forming most ofthe mass of the freeze-dried product (typically from 50 up to more than600 times the weight of the active components forming the stabilizinglayer of the microvesicles). For instance, PEG4000 has a meltingtemperature of 53-58° C. According to an embodiment, the heatingtemperature is preferably of 50° C. or lower. Preferred temperatures forthe heat treatment are from 38° C. to 45° C.

The duration of the heat treatment generally depends on the temperatureof the treatment; typically, the higher the temperature, the shorter theduration of the heating. As the materials forming the gas-filledmicrovesicles envelope (phospholipids in particular) may undergodegradation reaction if subjected to excessive temperatures for a toolong period of time (with possible negative consequences on thecharacteristics of the reconstituted microvesicles), the duration of theheat treatment shall not be unnecessarily prolonged. While a treatmentduration of about 8 hours may be sufficient (particularly in combinationwith temperatures higher than 45° C., e.g. 48° C.), the duration of theheat treatment is preferably performed for 12 hours, up to e.g. 20hours, more preferably 14 to 18 hours. While in particular cases longerdurations may well be applied (particularly in combination withtemperatures lower than 45° C., preferably lower than 42° C.), theApplicant has observed that the characteristics of the final gas-filledmicrovesicles are only slightly if not at all further improved; suchincreased duration is thus in most cases not necessary and generallyinconvenient in terms of manufacturing economy at the industrial scale.

In certain embodiments, the freeze-dried product comprises a mixture ofa phospholipid and of a fatty acid, as above defined, in admixture witha freeze-drying protecting component. The thermal treatment of theinvention has been proven to be particularly effective for improving thecharacteristics of gas-filled microvesicles comprising such mixture ofcomponents.

According to an embodiment, the freeze-dried product comprises DSPC,DPPG and palmitic acid in combination with a freeze-drying protectingcomponent (e.g. polyetheleneglycol, such as PEG4000). Said freeze-driedproduct is preferably heated at a temperature of from about 40° C. to48° C., particularly of about 45° C. (+/−3° C.) for at least eighthours, preferably for about 18 h (+/−4 h).

According to another embodiment of the invention, the freeze-driedproduct comprises DSPC, DPPE-PEG5000 and palmitic acid in combinationwith a freeze-drying protecting component (e.g. polyetheleneglycol, suchas PEG4000). Said freeze-dried product is heated at a temperature offrom about 36° C. to 45° C., particularly of about 39° C. (+/−3° C.) forat least eight hours, preferably for about 15 h (+/−5 h).

According to a further embodiment, said mixture of DSPC, DPPE-PEG5000and palmitic acid further comprises a targeting lipopeptide, e.g. asdescribed in WO2007/067979.

As mentioned above, the thermal treatment of the freeze-dried productaccording to the invention results in an increased resistance of thegas-filled microvesicles to pressure. Advantageously, microvesicles withincreased resistance to pressure generally show an increased timepersistency in the blood stream once injected.

Resistance to pressure of gas-filled microvesicles can be assessed bydetermining the empiric parameter “Pc50” or “critical pressure”.

As explained in detail in the experimental part, the Pc50 of asuspension of gas-filled microvesicles identifies the value of appliedoverpressure (with respect to atmospheric pressure) at which theabsorbance of a suspension of microvesicles drops to half of theabsorbance of the suspension measured at atmospheric pressure, saidapplied overpressure resulting in a substantial reduction of thepopulation of microvesicles with respect to the initial one (atatmospheric pressure). As a matter of fact, reduction of the absorbanceof a suspension of microvesicles is related to the reduction of theinitial population of gas-filled microvesicles, whereby the initiallymilky suspension (high concentration of microvesicles) becomes more andmore transparent under increasing pressure (reduced concentration due tocollapse of microvesicles). The higher the Pc50 values, the higher theresistance to pressure of microvesicles. For ultrasound diagnosticapplications, a minimum Pc50 value of at least 12 kPa is desirable forgas-filled microvesicles, preferably at least 13 kPa (about 100 mmHg),more preferably at least 14 kPa (105 mmHg). For ultrasound therapeuticapplications, generally needing longer persistency time in the bloodflow, a minimum Pc50 value of at least 55 kPa (about 412 mmHg) isdesirable, preferably at least 70 kPa (about 525 mmHg), more preferablyat least 80 kPa (about 600 mmHg), while higher values of Pc50 are evenmore preferred.

Typically, the thermal treatment of the freeze-dried product accordingto the invention allows increasing the Pc50 of the reconstitutedsuspension of microvesicles of at least 5 kPa, preferably at least 8 kPaand more preferably at least 10 kPa with respect to the Pc50 of areconstituted suspension obtained from a freeze-dried product which hasnot been submitted to such thermal treatment. Such increase of Pc50 maybe up to 15 kPa and in some embodiments up to 25 kPa.

According to an embodiment (e.g. when the freeze-dried product comprisesDSPC, DPPG, palmitic acid and PEG4000) a suspension of microvesiclesreconstituted from freeze-dried product subjected to a thermal treatmentaccording to the invention has a value of Pc50 of at least 20 kPa,preferably at least 22 kPa and more preferably of at least 25 kPa.

According to another embodiment (e.g. when the freeze-dried productcomprises DSPC, DPPE-PEG5000 and palmitic acid in combination with afreeze-drying protecting component, e.g. polyetheleneglycol, such asPEG4000) a suspension of microvesicles reconstituted from a freeze-driedproduct subjected to a thermal treatment according to the invention hasa value of Pc50 of at least at least 75 kPa, preferably at least 80 kPaand more preferably of at least 90 kPa.

Pharmaceutical Kit, Administration and Methods of Use

The vials containing the freeze-dried product can be advantageouslypackaged in a two component diagnostic and/or therapeutic kit,preferably for administration by injection. The kit preferably comprisesthe vial containing the freeze-dried product and a second container(e.g. a syringe barrel) containing the physiologically acceptableaqueous carrier for reconstitution.

The microvesicles of the present invention may be used in a variety ofdiagnostic and/or therapeutic techniques, including in particularultrasound.

An aspect of the invention thus relates to the use in a method ofdiagnosing of a suspension of microvesicles reconstituted fromfreeze-dried product subjected to a thermal treatment according to theinvention.

Diagnostic methods include any method where the use of the gas-filledmicrovesicles allows enhancing the visualisation of a portion or of apart of an animal (including humans) body, including imaging forpreclinical and clinical research purposes. A variety of imagingtechniques may be employed in ultrasound applications, for exampleincluding fundamental and harmonic B-mode imaging, pulse or phaseinversion imaging and fundamental and harmonic Doppler imaging; ifdesired three-dimensional imaging techniques may be used.

Microvesicles according to the invention may typically be administeredin a concentration of from about 0.01 to about 1.0 μL of gas per kg ofpatient, depending e.g. on their respective composition, the tissue ororgan to be imaged and/or the chosen imaging technique. This generalconcentration range may of course vary depending on specific imagingapplications, e.g. when signals can be observed at very low doses suchas in colour Doppler or power pulse inversion.

In an embodiment said method of diagnosing comprises

-   -   (i) administering to a patient a suspension of gas-filled        microvesicles obtained by reconstitution of a freeze-dried        product obtained according to the process of the invention; and    -   (ii) detecting an ultrasound signal from a region of interest in        said patient.

According to an embodiment, said suspension of gas-filled microvesiclescomprises DSPC, DPPG, palmitic acid and PEG4000.

Reconstitution of the freeze-dried product is preferably made bydispersing it into a physiologically acceptable aqueous carrier, e.g.saline, in the presence of a physiologically acceptable gas, e.g SF₆,under gentle agitation.

Said suspension of microvesicles has preferably a value of Pc50 of atleast 20 kPa, more preferably at least 22 kPa and even more preferablyof at least 25 kPa.

In an embodiment, said method of diagnosing comprises ultrasound imagingof the heart, in particular to opacify the left ventricular chamber andto improve the delineation of the left ventricular endocardial border inadult patients with suboptimal echocardiograms.

In another embodiment, said method of diagnosing comprises ultrasoundimaging of the liver, in particular for characterization of focal liverlesions in adult and pediatric patients.

In a further embodiment, said method of diagnosing comprises ultrasoundimaging of the urinary tract, particularly for the evaluation ofsuspected or known vesicoureteral reflux in pediatric patients.

In another embodiment of said diagnostic method, said suspension ofgas-filled microvesicles comprise DSPC, DPPE-PEG5000, palmitic acid,optionally a targeting lipopeptide and PEG4000. Preferably the targetinglipopeptide is a VEGF/KDR targeting lipopeptide, e.g. as described inWO2007/067979.

Possible other diagnostic imaging applications include scintigraphy,light imaging, and X-ray imaging, including X-ray phase contrastimaging.

Another aspect of the invention relates to the use in a method oftherapeutic treatment of a suspension of microvesicles reconstitutedfrom freeze-dried product subjected to a thermal treatment according tothe invention.

Therapeutic techniques include any method of treatment (as abovedefined) of a patient which comprises the combined use of ultrasoundsand gas-filled microvesicles either as such (e.g. in ultrasound mediatedthrombolysis, high intensity focused ultrasound ablation, blood-brainbarrier permeabilization, immunomodulation, neuromudulation,radiosensitization) or in combination with a therapeutic agent (i.e.ultrasound mediated delivery, e.g. for the delivery of a drug orbioactive compound to a selected site or tissue, such as in tumortreatment, gene therapy, infectious diseases therapy, metabolic diseasestherapy, chronic diseases therapy, degenerative diseases therapy,inflammatory diseases therapy, immunologic or autoimmune diseasestherapy or in the use as vaccine), whereby the presence of thegas-filled microvesicles may provide a therapeutic effect itself or iscapable of enhancing the therapeutic effects of the applied ultrasounds,e.g. by exerting or being responsible to exert a biological effect invitro and/or in vivo, either by itself or upon specific activation byvarious physical methods (including e.g. ultrasound mediated delivery).

Microvesicles according to the invention can typically be administeredfor therapeutic purposes in a concentration of from about 0.01 to about5.0 μL of gas per kg of patient, depending e.g. from their respectivecomposition, the type of subject under treatment, the tissue or organ tobe treated and/or the therapeutic method applied.

In an embodiment said method of ultrasound therapeutic treatmentcomprises:

-   -   (i) administering to a patient a suspension of gas-filled        microvesicles obtained by reconstitution of a freeze-dried        product obtained according to the process of the invention;    -   (ii) identifying a region of interest in said patient to be        submitted to a therapeutic treatment, said region of interest        comprising said suspension of gas-filled microvesicles; and    -   (iii) applying an ultrasound beam for therapeutically treating        said region of interest;

whereby said ultrasound therapeutic treatment is enhanced by thepresence of said suspension of gas-filled microvesicles in said regionof interest.

In an embodiment, said suspension of gas-filled microvesicles comprisesDSPC, DPPE-PEG5000, palmitic acid, optionally a targeting lipopeptideand PEG4000.

Reconstitution of the freeze-dried product is preferably made bydispersing it into a physiologically acceptable aqueous carrier, e.g.saline, in the presence of a physiologically acceptable gas, e.g. amixture of C₄F₁₀ and nitrogen, under gentle agitation.

Said suspension of microvesicles has preferably a value of Pc50 of atleast 84 kPa, more preferably at least 88 kPa and even more preferablyof at least 90 kPa, up to about e.g. 105 kPa.

In a further embodiment, the suspension of gas-filled microvesicles ofthe invention may be advantageously used in a method for separatingcells, typically by buoyancy (also known as buoyancy-activated cellsorting, “BACS”). The method can be useful for separating a desired typeof cells from other cells in a physiological liquid (e.g. blood orplasma). In an embodiment, the separation method comprises labelling adesired cell to be separated with a suitable labelled antibody capableof binding to a specific (and selective) receptor on said cell. Themicrovesicles of the invention are then added to the suspension of cellsto be separated (including those bearing the labelled antibody); onceadmixed to the suspension of cells, the microvesicles associate throughthe ligand with the labelling residue bound to antibody/cell constructthus allowing separation of the cells by buoyancy (see e.g. WO2017/117349). For instance, the labelled antibody is a biotinylatedantibody, where the biotin residue is capable of associating with arespective moiety, such as for instance an avidin, neutravidin orstreptavidin residue on a gas-filled microvesicles. The improvedresistance to pressure allows using the microvesicles of the inventionin a wide variety of methods for separating cells.

The following examples will help to further illustrate the invention.

EXAMPLES

Materials

DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine

DPPG-Na: 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodiumsalt)

DPPE-PEG5000:1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (ammonium salt)

PEG4000=Polyethylenglycol (MW=4000 g/mol)

Measurement of Pressure Resistance (Pc50)

The resistance to pressure of gas-filled microvesicles was evaluatedusing an in-house developed pressure nephelometer. Briefly, themicrovesicles suspension was introduced into a spectrophotometer samplecell (airtight and connected to a pressurization system). The opticaldensity (absorbance at 700 nm) of the suspension is continuouslyrecorded while linearly increasing the pressure applied to the sample inthe cell from atmospheric pressure (760 mmHg, 101.3 kPa,) to an overpressure of two bars (2280 mmHg, 303.9 kPa), at a rate of about 4 mmHg/s(533 Pa/s).

The Pc50 parameter (“critical pressure”) characterizing each suspensionidentifies the overpressure (with respect to atmospheric pressure) atwhich the absorbance of the microvesicles suspension drops to half ofits initial value.

Example 1

Preparation of Freeze-Dried Product (Batches 1a-1b)

The procedure illustrated in the working examples of WO 94/09829 wasused for preparing two different batches (1a-1b) each consisting ofseveral vials containing the freeze-dried product.

Briefly, DSPC, DPPG-Na and palmitic acid in a weight ratio of4.75/4.75/1 were first dissolved in hexane/ethanol (8/2, v/v) at aconcentration of about 5 g/L and the solvents were evaporated undervacuum. The solid residue was admixed with PEG4000 in a weight ratio ofabout 0.017:1, the mixture was dissolved in tert-butanol at around 60°C. and the clear solution was used to fill respective DIN8R vials (witha corresponding volume containing about 25 mg of the mixture). The vialswere then rapidly cooled at −45° C. and then subjected the vacuum forremoving the frozen solvent by sublimation. The temperature was thenraised (above room temperature, not higher than 35° C.) and theremaining solvent was evaporated, down to a final amount of less than0.5% by weight. At the end of the freeze-drying process, the ambient ofthe freeze-dryer was saturated with SF₆ at atmospheric pressure and thevials (containing the solid freeze-dried product in contact with SF₆)were stoppered and sealed.

The two batches were used for the subsequent heat treatment experiments.

Example 2

Preparation of Freeze-Dried Product (2a-2h)

The procedure illustrated in the working examples of WO2004/069284 wasused for preparing eight different batches (2a-2h) each consisting ofseveral vials containing the freeze-dried product.

Briefly, an emulsion of cyclooctane and water (about 1.5/100 v/v)containing about 90 mg/I of DSPC, 7 mg/I of palmitic acid, 60 mg/I ofDPPE-PEG5000 and 100 g/l of PEG4000 is prepared (Megatron MT3000,Kinematica; 10′000 rpm) and sampled into DIN8R vials (about 1 ml/vial).

The vials were cooled at −50° C. under vacuum and then subjected tolyophilization, followed by secondary drying above room temperatureuntil complete removal of water and solvent (less than 0.5% by weight),as described in example 1. At the end of the freeze-drying process, theheadspace of the vials is saturated with a 35/65 mixture of C₄F₁₀/N₂ andthe vials are stoppered and sealed.

The different batches (1a to 1h) were used for the subsequent heattreatment experiments.

Example 3

Effect of the Heat Treatment on Batches Manufactured According toExample 1

The vials of the various batches prepared according to examples 1 weresubmitted to different heat treatments and the effect on thecharacteristics of the reconstituted suspensions of gas-filledmicrovesicles were observed.

Experiment 3.1

The vials of batch 1a were submitted to a heating temperature of 48° C.for a time ranging from 8 hours to one week (five vials for each group).The product in the vial was then reconstituted with 5 ml of saline andthe characteristics of the microvesicles in the suspension weremeasured. Results are reported in the following table 1.

TABLE 1 Batch 1a Heating time Pc50 (kPa) 48° C. mean value No heating14.4  8 hours 24.8 16 hours 25.5 24 hours 28.7 48 hours 28.8 One week32.0

As inferable from the above results, a substantial increase in thepressure resistance can be observed after 8 hours of heat treatment,with respect to the untreated freeze-dried samples. Such pressureresistance slightly increases in time, up to a maximum after one week oftreatment. However, as observed by the Applicant, a too long heatingtime (e.g. after 24 hours and particularly above 48 hours) maynegatively impact on other characteristics of the gas-filledmicrovesicles, such as their total number, the total volume of gasand/or their mean size.

Experiment 3.2

In a second experiment, the vials of batch 1b were heated attemperatures of 40° C., 45° C. and 49° C. for time periods of 12, 16 or20 hours (three vials for each group, for a total of 27 vials). Theproduct in the vial was then reconstituted with 5 ml of saline and thecharacteristics of the microvesicles in the suspension were measured.Results are reported in the following table 2.

TABLE 2 Batch 1b Pc50 (kPa) Heating time T (° C.) mean value 12 hours 4022.8 16 hours 40 23.6 20 hours 40 23.7 12 hours 45 25.5 16 hours 45 22.520 hours 45 22.4 12 hours 49 24.1 16 hours 49 24.4 20 hours 49 24.9

As inferable from the above data, a substantial increase in the pressureresistance is obtained with the heat treatment of the freeze-driedproducts (with respect to the initial value of about 14 kPa). While ahigher increase of Pc50 may generally be observed for treatments at 49°C., treating at this temperature may however negatively impact on othercharacteristics of the reconstituted gas-filled microvesicles,particularly on the mean size values.

Example 4

Effect of the Heat Treatment on Batches Manufactured According toExample 2

The vials of the various batches (2a-2h) prepared according to example 2were submitted to different heat treatments and the effect on thecharacteristics of the reconstituted suspensions of gas-filledmicrovesicles were observed.

Experiment 4.1

The vials of batch 2a were submitted to a heating temperature of 40° C.or 45° C. for 16 hours or not heated. The product in the vial was thenreconstituted with 5 ml of saline and the characteristics of themicrovesicles in the suspension were measured. Results are reported inthe following table 3.

TABLE 3 Batch 1a Heating T Pc50 (kPa) for 16 h mean value No heating66.1 40° C. 84.8 45° C. 78.8

As inferable from the above data, a substantial increase in the pressureresistance is obtained upon heat treatment also for batches manufacturedaccording to the procedure of example 2.

Experiment 4.2

The vials of batch 2b were submitted to a heating temperature of 40° C.for a time ranging from 16 to 88 hours, or not heated. The product inthe vial was then reconstituted with 5 ml of saline and thecharacteristics of the microvesicles in the suspension were measured.Results are reported in the following table 4.

TABLE 4 Batch 2b Heating time Pc50 (kPa) T = 40° C. mean value Noheating 82.1 16 hours 99.0 40 hours 103.6 64 hours 98.6 88 hours 102.5

As inferable from the above data, a substantial increase in the pressureresistance is obtained upon heat treatment at 40° C. A duration of thetreatment of 16 h is generally considered sufficient, also for avoidingpossible negative effects caused by longer thermal treatments on othercharacteristics of the microvesicles (e.g. increase of large sizemicrovesicles in the reconstituted suspension).

Experiment 4.3

The vials of batches 2c-2g were submitted to a heating temperature of40° C. for a period of 16 hours, or not heated. The product in the vialwas then reconstituted with 5 ml of saline and the characteristics ofthe microvesicles in the suspension were measured. Results are reportedin the following table 5.

TABLE 5 Batches 2c-2g (40° C., 16 h) Thermal Pc50 (kPa) Batch No.Treatment mean value 2c No 70.6 2c Yes 93.7 2d No 74.1 2d Yes 94.3 2e No69.6 2e Yes 94.2 2f No 62.8 2f Yes 79.8 2g No 55.4 2g Yes 81.3

As inferable from the above table, for the suspensions of microvesiclesreconstituted from the various batches an increase in pressureresistance of more than 15 kPa or more and up to about 25 kPa isobtained after heat treatment of the freeze-dried products.

Experiment 4.4

The vials of batch 2h were submitted to a heat treatment at 38° C. for atime ranging from two to 24 hours. The product in the vial was thenreconstituted with 5 ml of saline and the characteristics of themicrovesicles in the suspension were measured. Results are reported inthe following table 6.

TABLE 6 Batch 2h Heating time Pc50 (kPa) (h) at 38° C. mean value 063.19 2 73.33 4 74.66 6 79.33 8 80.26 12 82.66 16 79.73 24 83.06

As inferable from the above table, an increasing pressure resistance ofthe microvesicles in the reconstituted suspension is obtained uponheating the freeze-dried material for an increasing time, up to 8-12hours at 38° C. Further heating of the material (16 or 24 hours) doesnot substantially further increase the pressure resistance.

The invention claimed is:
 1. A method of manufacturing a freeze-driedcomposition suitable for the preparation of a suspension of stabilizedgas-filled microbubbles, said composition comprising: (i) an amphiphilicmaterial comprising a phospholipid and a fatty acid; and (ii) apolyethylene glycol as freeze-drying protecting component; whichcomprises: a. preparing a liquid mixture comprising said amphiphilicmaterial and said freeze-drying protecting component in a solvent; b.freeze-drying the liquid mixture to remove said solvent and obtain afreeze-dried product; and c. after completion of the freeze-drying ofstep b, heating said freeze-dried product at ambient pressure at atemperature higher than 35° C. and lower than the melting point of thepolyethylene glycol freeze-drying protecting component, for a period oftime of from eight to twenty hours, wherein the freeze-dried product ofstep b has not been reconstituted prior to step c.
 2. The methodaccording to claim 1 wherein said liquid mixture comprises saidamphiphilic material and said freeze-drying protecting componentdispersed in an organic solvent.
 3. The method according to claim 1wherein said liquid mixture comprises said amphiphilic material and saidfreeze-drying protecting component dispersed in an aqueous emulsion of awater immiscible solvent and water.
 4. The method according to claim 1wherein said heating step is carried out at a temperature of from 38° C.to the melting point of the polyethylene glycol freeze-drying protectingcomponent.
 5. The method according to claim 1 wherein said heating stepis carried out at a temperature of from 40° C. to the melting point ofthe polyethylene glycol freeze-drying protecting component.
 6. Themethod according to claim 1 wherein said phospholipid comprisesdilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine(DMPC), dipalmitoyl-phosphatidylcholine (DPPC),diarachidoyl-phosphatidylcholine (DAPC), distearoyl-phosphatidylcholine(DSPC), dioleoyl-phosphatidylcholine (DOPC), 1,2Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC),dipentadecanoyl-phosphatidylcholine (DPDPC),1-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC),1-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC),1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC),1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC),1-palmitoyl-2-oleylphosphatidylcholine (POPC),1-oleyl-2-palmitoyl-phosphatidylcholine (OPPC),dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts,diarachidoylphosphatidyl-glycerol (DAPG) and its alkali metal salts,dimyristoylphosphatidylglycerol (DMPG) and its alkali metal salts,dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts,distearoylphosphatidylglycerol (DSPG) and its alkali metal salts,dioleoyl-phosphatidylglycerol (DOPG) and its alkali metal salts,dimyristoyl phosphatidic acid (DMPA) and its alkali metal salts,dipalmitoyl phosphatidic acid (DPPA) and its alkali metal salts,distearoyl phosphatidic acid (DSPA), diarachidoylphosphatidic acid(DAPA) and its alkali metal salts, dimyristoyl-phosphatidylethanolamine(DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidyl-ethanolamine (DSPE), dioleylphosphatidyl-ethanolamine(DOPE), diarachidoylphosphatidylethanolamine (DAPE),dilinoleylphosphatidylethanolamine (DLPE), dimyristoylphosphatidylserine (DMPS), diarachidoyl phosphatidylserine (DAPS),dipalmitoyl phosphatidylserine (DPPS), distearoylphosphatidylserine(DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoyl sphingomyelin(DPSP), and di stearoyl sphingomyelin (DSSP),dilauroyl-phosphatidylinositol (DLPI), diarachidoylphosphatidylinositol(DAPI), dimyristoylphosphatidylinositol (DMPI),dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol(DSPI) or dioleoyl-phosphatidylinositol (DOPI).
 7. The method accordingto claim 1 wherein said fatty acid comprises capric (n-decanoic), lauric(n-dodecanoic), myristic (n-tetradecanoic), palmitic (n-hexadecanoic),stearic (n-octadecanoic), arachidic (n-eicosanoic), behenic(n-docosanoic) or n-tetracosanoic acid.
 8. The method according to claim1, wherein said amphiphilic material comprises DSPC, DPPG and palmiticacid.
 9. The method according to claim 1, wherein said amphiphilicmaterial comprises DSPC, DPPE-PEG5000 and palmitic acid.
 10. The methodaccording to claim 8, wherein said heating step is carried out at atemperature of from 40° C. to 48° C.
 11. The method according to claim9, wherein said heating step is carried out at a temperature of from 36°C. to 45° C.
 12. The method according to claim 1, wherein said heatingin step c is performed for a period of time of from twelve to twentyhours.
 13. The method according to claim 1 wherein said liquid mixtureis sampled into glass vials which are loaded into a freeze-dryer. 14.The method according to claim 13 which comprises, at the completion ofstep b, saturating the headspace of the vials containing thefreeze-dried product with a physiologically acceptable gas and thenstoppering and sealing the vials.
 15. A method of manufacturing asuspension of gas-filled microvesicles comprising (i) preparing a liquidmixture comprising an amphiphilic material and a freeze-dryingprotecting component in a solvent, wherein the amphiphilic materialcomprises a phospholipid and a fatty acid and the freeze-dryingprotecting component is a polyethylene glycol; (ii) freeze-drying theliquid mixture to remove said solvent and obtain a freeze-dried product;(iii) after completion of the freeze-drying of step (ii), heating saidfreeze-dried product at ambient pressure at a temperature higher than35° C. and lower than the melting point of the polyethylene glycolfreeze-drying protecting component, for a period of time from eight totwenty hours, wherein the freeze-dried product of step (ii) has not beenreconstituted prior to step (iii); and (iv) reconstituting saidfreeze-dried product with a pharmaceutically acceptable liquid carrierin the presence of a physiologically acceptable gas under gentleagitation to obtain a suspension of gas-filled microvesicles.
 16. Themethod of claim 15, wherein the physiologically acceptable gas isselected from SF₆, C₃F₈, and C₄F₁₀, optionally in admixture with air ornitrogen.
 17. The method of claim 15, wherein the phospholipid consistsof the combination of DSPC and the sodium salt of DPPG, and the fattyacid consists of palmitic acid.
 18. The method of claim 15, wherein thephospholipid consists of the combination of DSPC and DPPE-PEG5000 andthe fatty acid consists of palmitic acid.
 19. A method comprising: (i)preparing a liquid mixture comprising an amphiphilic material and afreeze-drying protecting component in a solvent, wherein the amphiphilicmaterial comprises a phospholipid and a fatty acid; and thefreeze-drying protecting component is a polyethylene glycol; (ii)freeze-drying the liquid mixture to remove said solvent and obtain afreeze-dried product; (iii) after completion of the freeze-drying ofstep (ii), heating said freeze-dried product at ambient pressure at atemperature higher than 35° C. and lower than the melting point of thepolyethylene glycol freeze-drying protecting component, for a period oftime from eight to twenty hours, wherein the freeze-dried product ofstep (ii) has not been reconstituted prior to step (iii); and (iv)reconstituting said freeze-dried product with a pharmaceuticallyacceptable liquid carrier in the presence of a physiologicallyacceptable gas under gentle agitation in order to obtain a suspension ofgas-filled microvesicles; (v) administering to a patient said suspensionof gas-filled microvesicles; and (vi) detecting an ultrasound signalfrom a region of interest in said patient.
 20. The method according toclaim 19, wherein the region of interest in said patient is the heart.21. The method according to claim 19, wherein the region of interest insaid patient is the liver.
 22. The method according to claim 19, whereinthe region of interest in said patient is the urinary tract.
 23. Themethod of claim 19, wherein the physiologically acceptable gas isselected from SF₆, C₃F₈, and C₄F₁₀, optionally in admixture with air ornitrogen.
 24. The method of claim 19, wherein the phospholipid consistsof the combination of DSPC and the sodium salt of DPPG, and the fattyacid consists of palmitic acid.
 25. The method of claim 19, wherein thephospholipid consists of the combination of DSPC and DPPE-PEG5000 andthe fatty acid consists of palmitic acid.